The research program described in this application deals with the mechanism of copper-responsive gene expression. The experimental model is the green alga Chlamydomonas reinhardtii which is ideally suited for studies of copper and copper-protein metabolism. Previous work has shown that the accumulation of several proteins in this organism is dependent upon the concentration of copper in the medium. One of these is plastocyanin, an abundant copper- containing photosynthetic electron transfer catalyst, whose copper- dependent accumulation is effected by metal-specific holoprotein formation and increased stability of the copper-protein relative to the apoprotein. Another is cytochrome C6 (a heme containing substitute electron transfer protein), whose synthesis is induced in copper-deficient cells by transcriptional activation of its gene mediated -a copper-responsive elements (CuREs) and a CuRE-binding protein (BP). A third is the enzyme coproporphyrinogen (coprogen) oxidase, a catalyst of a late step in the heme biosynthetic pathway, which is induced coordinately with cyt c6 in copper- deficient cells. The objective of the research planned for the next project period is to identify and characterize biochemically and by reverse genetics the components of the copper-dependent transcriptional response in this system, and to elucidate the molecular basis for some of its key features, particularly the sensitivity and selectivity of the metalloregulator with respect to its metal- and DNA-binding site(s). Emphasis will be placed on the CuRE-BP which will be purified on the basis of its sequence specific binding to previously identified CuREs associated with the cyt c6 gene. Biochemical and biophysical analyses will be employed to understand the structural basis of its copper-responsive function. cDNA and genomic sequences encoding the CuRE-BP will be cloned by conventional methods. In parallel experiments, "tagged' regulatory mutants will be identified by screening colonies of insertionally- mutagenized cells for loss of copper-responsive expression of a reporter gene. The wild-type alleles of the tagged genes will be cloned and expressed so that a reverse genetic approach can be exploited for the functional analysis of the metalloregulator. A second aim is the isolation and molecular characterization of copper responsive elements on the coprogen oxidase gene with the objective of determining whether a common regulatory pathway serves several copper-responsive genes and, if so, whether the CuRE-BP displays preferential utilization of CuREs on different genes. The long term objective of this research program is the understanding of copper homeostasis in eukaryotic cells, specifically the regulation of gene expression by copper in the context of adaptation to copper deficiency. The proposed project will expand our knowledge of the response mechanisms by which organisms allocate nutritionally-limited resources so as to maintain metabolically-essential functions, and in so doing, will contribute to the understanding of human copper imbalance which can result from reduced intake, excessive excretion or genetic diseases.